Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation.
ABSTRACT Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.
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ABSTRACT: The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants. We show that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation. We also show that this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants. The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. The data also suggest that the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.Current Biology 04/1996; 6(3):325-30. · 9.49 Impact Factor
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ABSTRACT: A method for Agrobacterium-mediated transformation of Kalanchoe blossfeldiana Poelln. (Crassulaceae) and quantitative analysis of the expression pattern of β-glucuronidase (GUS) gene are reported. Leaf segments were infected with the Agrobacterium strain LBA4404 having a binary vector plasmid pBI121 or its derivatives and cultured on Murashige and Skoog medium supplemented with 0.5 mg/l benzyladenine, 2 mg/l indoleacetic acid, 300 mg/l carbenicillin and 20 mg/l kanamycin (selection medium) for regeneration. Primary transformants (R0) with three kinds of chimeric GUS genes were obtained in different experiments, and Southern blot analysis confirmed the presence of the GUS gene in their genome. The integrated GUS gene(s) seemed to be silenced in about half of the transformants. There was no clear correlation between copy number and expression level of the GUS gene. Observed levels of GUS activity within transformants were divided into three classes: (1) high activity, (2) low activity and (3) near-zero activity. Selfed progeny (R1) of a near-zero activity and a low activity transformant segregated to high, low and near-zero activity. Southern blot analysis unexpectedly revealed no clear difference in the band pattern of the GUS gene; some progeny showed high activity while some progeny showed near-zero activity derived from the near-zero-activity transformant.Plant Science - PLANT SCI. 01/1996; 121(2):175-185.
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ABSTRACT: Reduction in the amounts of active gibberellic acids (GA) in elongating cuttings from the ornamental crop Kalanchoe blossfeldiana were pursued by genetic manipulation as an alternative to synthetic growth regulators. An alcohol inducible promoter system was used to control silencing of GA activating enzymes. Apart from affecting the stem length, abnormal levels of GA can lead to altered flowering time, lacking seed maturation and changes in morphology. The effects of down regulating a group of GA 20-oxidases were investigated in fast growing cuttings of K. blossfeldiana Poelln. cv. Molly. The transgenic plants were phenotypically indistinguishable from wild type plants until silencing was induced by low concentrations of ethanol. Treated plants were reduced in height but otherwise appeared normal; flowering was delayed but with large variations in time between the transgenic lines. These data indicate that optimisation of the ethanol treatments can enable us to produce more compact growing plants still maintaining normal flowering.Plant Cell Tissue and Organ Culture 05/2008; 93(3):241-248. · 3.63 Impact Factor